Evaluation of intracellular lipids by standardized staining with a Sudan black B fraction.

The selective staining of neutral lipids in Human Amnion cells in tissue culture was achieved using a particular fraction of the lipid dye, Sudan black B and a standardized staining procedure. The fraction, termed SBB-I, was isolated by thin-layer chromatography. The cytophotometric assessment of intracellular neutral lipids, stained with SBB-I, is described and applied to the study of changes in granulocytic neutral lipids in leukemia.     

Some aspects of the value of Sudan Black B in lipid histochemistry

Summary The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films.Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (21) before stainig resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lambert-Beer. Proteins were however, also coloured by the dye.The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show aspecific binding.The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.     

Some aspects of the value of Sudan Black B in lipid histochemistry.

The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films. Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (2:1) before staining resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lamber-Beer, Proteins were however, also coloured by the dye. The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show a specific binding. The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.     

Evidence against the existence of acetylated Sudan Black B

Summary The nature of acetylated Sudan Black B (aSBB) has been investigated, and it has been found, by thin layer chromatography, that each fraction of aSBB has an R _f which is the same as that of a similar fraction of Sudan Black B (SBB). However, aSBB has been found to have fewer fractions, 9–12 than SBB, 14–16. The two major fractions from aSBB and SBB were examined, and a great similarity was found between the absorption spectra of the respective fractions of aSBB and SBB. The major fraction of aSBB was investigated by mass spectroscopy and found to have a similar molecular weight to that expected of SBB. This demonstrates that aSBB is not in fact acetylated, and that the components of aSBB are chemically no different from the corresponding components of SBB.     

The value of simple lipid stains for typing skeletal muscle fibres

Summary Skeletal muscle fibre types can be distinguished rapidly with simple lipid stains. Comparative studies showed that Sudan Black B is superior to Oil Red O for this purpose and that optimum staining is obtained using unfixed sections or sections fixed in calciumglutaraldehyde. Factors that possibly influence the staining reaction, such as freeze-thawing, are considered. The stained lipids were identified by thin layer chromatography.     

Evidence against the existence of acetylated Sudan Black B

The nature of acetylated Sudan Black B (aSBB) has been investigated, and it has been found, by thin layer chromatography, that each fraction of aSBB has an Rf which is the same as that of a similar fraction of Sudan Black B (SBB). However, aSBB has been found to have fewer fractions, 9-12 than SBB, 14-16. The two major fractions from aSBB and SBB were examined, and a great similarity was found between the absorption spectra of the respective fractions of aSBB and SBB. The major fraction of aSBB was investigated by mass spectroscopy and found to have a similar molecular weight to that expected of SBB. This demonstrates that aSBB is not in fact acetylated, and that the components of aSBB are chemically no different from the corresponding components of SBB.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

CHANGE OF FATTY ACID COMPOSITION OF CHLORELLA ELLIPSOIDEA DURING ITS CELL CYCLE

The lipids extracted from Chlorella cells at different developmentalstages were separated by chromatography on silicic acid into"nonpolar" (chloroform-eluate) and "polar" (methanol-eluate)lipid fractions. The lipids were also subjected to florisilchromatography to fractionate neutral glycerides and free fattyacids. Gas-liquid chromatographic analysis of these fractions has revealeda marked difference in their fatty acid compositions which werefound to undergo characteristic changes during the course ofalgal cell cycle. It was found that the fatty acids in the "nonpolar"lipid (fat) fraction are synthesized during the growth phasein the light and consumed during the process of cellular division. (Received September 24, 1966; )     

Arachidonic and docosahexanoic acid content of bovine brain myelin: Implications for the pathogenesis of multiple sclerosis

Lipids were extracted from bovine brain myelin using a mixture of hexane and isopropanol (32). Myelin lipids were resolved, using Sep Pak chromatography, into four fractions: Fraction 1 contained neutral lipids, fraction 2, free fatty acids, fraction 3, ethanolamine phospholipids and fraction 4, choline phospholipids. Docosahexanoic (DHA) and arachidonic (AA) acids in these fractions were measured by RPHPLC. Fraction 2 was analyzed directly, the other three fractions were subjected to alkaline hydrolysis before analysis for DHA and AA. DHA and AA were not found in fraction 1. Both DHA and AA were found in fractions 2 and 3. Only AA was consistently found in fraction 4. These results were confirmed by GC.     

Studies on the Lipid Components of Endotoxin II. Chemical Analyses and Biological Properties of Neutral,Polar-I and Polar-II Subfractions

Lipid components obtained from Salmonella typhosa O-901 endotoxin by acid hydrolysis were separated into neutral, polar-I and polar-II lipid fractions by silica gel column chromatography. These lipids were further separated by silica gel column and/or thin-layer chromatography. The subfractions were analyzed by thin-layer chromatography, gas chromatography and infrared spectrophotometry. Seven subfractions obtained from the neutral lipid fraction contained lauric, myristic, palmitic, 3-OH-myristic acid, artificial products of 3-OH-myristic acid, or a small amount of two unidentified fatty acids. These fatty acids and glucosamine were commonly detected in six subfractions obtained from the polar-I lipid fraction. Fatty acids, glucosamine, and O-phosphorylethanolamine were detected in all of the 13 subfractions obtained from the polar-II lipid fraction. Chick embryo lethal activity, rabbit pyrogenicity and in vitro interferon inducing activity were found in three polar-I lipid subfractions and five polar-II lipid subfractions, but not in neutral lipids. The activities were highest in a polar-II lipid subfraction, which contained smaller amounts of O-phosphorylethanolamine and glucosamine than the other subfractions. However, no particular chemical constituent (s) related to the biological activities could be found. Prolonged acid hydrolysis of the polar-II lipids gave rise to neutral and polar-I lipids. Chemical and biological aspects of the lipid constituents of endotoxin are discussed.     

Zur Physikochemie der vitalen Kern- und Plasmafluorochromierung vonAllium cepa-Epidermen

Summary Microfluorimetrically recorded fluorescence bands of vitally stained nuclei and protoplasts from the inner epidermis of yellow onion(Allium cepa) scales are compared with fluorescence bands of Tarions model solutions. The strong fluorescent staining of the nucleus and the weaker hue of the cytoplasm after application of acridine orange is mainly due to the accumulation of monomeric dye cations in polar cytoplasmic lipids. Vital fluorescent staining with neutral red shows similar effects, but in addition an accumulation of the dye base in apolar lipids can be ascertained without doubt. On the other hand, the major accumulation of the acid fluorochrome uranin (sodium fluorescein) does not take place in polar cytoplasmic lipids but, apparently, rather in the form of anions in the water phase of the ground cytoplasm and inner nuclear plasm owing to a mechanism of plasmatic ion trap.The proof that dye ions are present in vitally stained protoplasm suggests the possibility, that the familiar phenomenon of vacuole contraction may depend on a Donnan effect in the case of basic dyes and might be a consequence of the raising of osmotic values by dye anions in the case of acid dyes.Experiments with three phases (dye solution-oil-blood plasma) yield fluorescent stainings of blood plasma with acridine orange, neutral red and uranin, which may be compared to vital staining of nuclei and cytoplasm. The well-known properties of blood plasma-lipids suggest a similar function of cytoplasmic lipids, viz. as a vehicle of lipid transport in a mainly aquatic, molecular-disperse phase. A diagram (Fig. 15) illustrates the cooperation between sheetlike lipoproteid complexes (boundary layers) and lipoproteid complexes of the ground cytoplasm dispersed as particles.     

The Lipid and Protein Staining Properties of Sudan II, III and IV and Their Components

Saturated solutions of commercial Sudan II, III and IV in 60% ethanol were found to stain heat-coagulated fat-free purified human serum albumin and defatted whole serum. The protein staining components were isolated and identified by means of paper chromatography on mineral oil-impregnated filter paper with 95% ethanol as the developing agent. The brownish and yellowish fractions which migrated rapidly in this system stained proteins well, whereas the red, pink and orange fractions, which migrated slowly, stained lipids only. The solubility and staining power of the slowly migrating lipid coloring fractions remained satisfactory in absence of the rapidly moving protein staining fractions.     

HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.     

Diurnal changes in liver and plasma lipids of choline-deficient rats

Early effects of choline deficiency were studied in rats. Nonphospholipid ("neutral lipid") and phospholipid were measured in plasma and in three fractions of a liver homogenate: sediment, supernatant fraction, and "floating fat." A single choline-deficient meal caused significant aberrations from the typical diurnal changes observed in the lipid fractions of the controls. These changes occurred in the following sequence: (a) failure of phospholipid to increase, after feeding, in the sediment fraction; (b) increase of neutral lipid, compared with controls, exclusively in the floating fraction; and (c) failure of neutral lipid to return to control levels. The rate of accumulation of neutral lipid increased during the first 4 days of deficiency. The occurrence of NADH-cytochrome c dehydrogenase in the floating fat and the absence of succinate dehydrogenase activity point to microsomal origin of the floating fat. Early effects of choline deficiency on plasma lipids were limited to phospholipid, and occurred later than changes in the liver. Plasma nonphospholipid levels were unchanged during the first 2 days; this does not support impaired secretion or transportation of glyceride as the cause of fatty liver in the early stages of choline deficiency.     

Chromatography and Biological Stains: III. Comparison of the Fat Staining Efficiency of Fractions of Commercial Sudan III Separated by Column Chromatography

Fractions of commercial Sudan III which were separated by column chromatography were compared for fat staining efficiency. Paraffin sections of chromated mouse liver tissue and sections of both fresh and formalin-fixed rat liver tissue or Musca domestica larvae, cut with the freezing microtome, were used. Evidence is presented that a sample of very highly purified Sudan III has no ability to render a fat stain in fresh, formalin-fixed or chromated tissue. However, certain other fractions from the commercial sample, some completely devoid of Sudan III, had good staining characteristics. It is concluded that some substance or substances, other than Sudan III, is responsible for the staining action of the commercial dye.     

Symbiodinium genotypic and environmental controls on lipids in reef building corals

Background

Lipids in reef building corals can be divided into two classes; non-polar storage lipids, e.g. wax esters and triglycerides, and polar structural lipids, e.g. phospholipids and cholesterol. Differences among algal endosymbiont types are known to have important influences on processes including growth and the photobiology of scleractinian corals yet very little is known about the role of symbiont types on lipid energy reserves.

Methodology/Principal Findings

The ratio of storage lipid and structural lipid fractions of Scott Reef corals were determined by thin layer chromatography. The lipid fraction ratio varied with depth and depended on symbiont type harboured by two corals (Seriatopora hystrix and Pachyseris speciosa). S. hystrix colonies associated with Symbiodinium C1 or C1/C# at deep depths (>23 m) had lower lipid fraction ratios (i.e. approximately equal parts of storage and structural lipids) than those with Symbiodinium D1 in shallow depths (<23 m), which had higher lipid fraction ratios (i.e. approximately double amounts of storage relative to structural lipid). Further, there was a non-linear relationship between the lipid fraction ratio and depth for S. hystrix with a modal peak at ∼23 m coinciding with the same depth as the shift from clade D to C types. In contrast, the proportional relationship between the lipid fraction ratio and depth for P. speciosa, which exhibited high specificity for Symbiodinium C3 like across the depth gradient, was indicative of greater amounts of storage lipids contained in the deep colonies.

Conclusions/Significance

This study has demonstrated that Symbiodinium exert significant controls over the quality of coral energy reserves over a large-scale depth gradient. We conclude that the competitive advantages and metabolic costs that arise from flexible associations with divergent symbiont types are offset by energetic trade-offs for the coral host.     

The acridine dyes: Their purification,physicochemical, and cytochemical properties

Summary The present investigation was designed to allow a critical comparison of the dye purity of six commerical acriflavine samples. Thin layer chromatography, absorption-, IR- and NMR-spectroscopy were applied for the identification of dye components and impurities. Ambiguities regarding the purity of the acriflavine samples have been resolved, showing that: (a) The finding permits the conclusion, that all analyzed samples of the fluorochrome acriflavine are characterized by a two-component dye pattern (acriflavine II and proflavine III), and contain fluorescent impurities. (b) The dye component III was the main component of only one dye sample.The effectiveness of these experiments is concerned with making automated microfluorometric measurement of cells stained with pure dye fractions more quantitative and reproduceable.This investigation was supported by a grant from the Bundesministerium für Forschung und Technologie (01 VH 065)     

Peroxidase and pseudoperoxidase reactions in relation to sudanophilia

Summary A selective staining of hemoglobin in erythroid cell series was achieved by use of Sudan Black B (modified method of Sheehan and Storey) if optimal amount of hydrogen peroxide was added to the staining mixture. The effect of some inhibitory agents (KCN, wet heat, pH) on this staining as well as on the Lepehne's pseudoperoxidase reaction for hemoglobin was similar. Both reactions were more resistant to these factors than the peroxidase reactions and sudanophilia in granulocytes in which both could be blocked by the pretreatment with absolute methanol. Moreover the effect of some extraction procedures for lipids on both myeloperoxidase reactions and sudanophilia was investigated. The results support the view that the sudanophilia in granulocytes is due to their peroxidase activity and for the staining of hemoglobin by use of Sudan Black B with H₂O₂ its pseudoperoxidase activity is responsible. In addition the effect of the substitution of phenolphosphate by dihydroxybenzenes on granulocyte sudanophilia is reported.     

Lipids of Pinus halepensis pollen

The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.     

Detection of lipids in the plant meristematic cell with the aid of Sudan black staining

As lipids can be a source of artefacts during intracellular localization of enzymes by cytochemical methodsin situ it was the aim of the present work to obtain orientation data on the distribution of lipids in the meristematic plant cells. The different fixation and object embedding methods examined revealed that it is best to fix the material by some formol fixative and without chroming, to embed it in polyethyleneglycol media. An alcoholic solution of Sudan black was found to be most reliable. In the meristematio cells the cytoplasm is usually stained more intensely than the nucleus. The ground cytoplasm is stained weakly while cytoplasmic particles are stained intensely. In some cases an intense black staining of nuclei, particularly in the prolongation zone, can be achieved. The staining intensity of cell components does not decrease on extracting lipids with pyridine. After extracting the dye from stained cell components a browninsh residual coloration remains. Chromatography of Sudan black revealed in all the samples tested slowly moving spots (blue and violet) and rapidly moving ones (red II, yellow, red I, colourless).